1. Mark the smears on the underside.
2. Place 1 to 2 loopfuls of water on the slide. 3. Transfer a small amount of culture with a sterile loop. (Mix with water) 4. Allow the smear to dry at room temperature or heat fix. 5. Cover with methylene blue for 30 seconds and rinse. Blot dry.

---FROM BROTH:

1. Mark the smears on the underside.
2. Place 2 to 3 loopfuls of the liquid culture on the slide with a sterile loop. 3. Spread the bacteria within the circle.
4. Allow the smear to dry at room temperature or heat fix. 5. Cover with methylene blue for 30 seconds and rinse. Blot dry.

NOTES:
Staphylococcus epidermisStaph- ClustersSmall blue circles

Bacillus megateriumStrep- ChainsBlue rods

What is the purpose of fixing a smear?It denatures bacterial enzymes. What would happen if too much heat were applied?Cells shapes would distort.

BMSC
Negative Stain

CULTURES: Bacillus subtilusStaphylococcus epidermis

MATERIALS: Nigrosin, Clean slide, Distilled water, Sterile toothpicks

FUNCTION: This technique is very useful in situations where other staining techniques do not show clear morphology or size.

PROCEDURE: 1. Place a small drop of nigrosin near on end of the slide. Mix a loopful of broth culture in the drop. (When from a solid medium, add a loopful of water) 2. Draw a second slide across the surface of the first until it contacts the drop. The drop will spread evenly across the edge of the top slide. 3. Push the top slide to the left along the entire surface of the bottom slide. 4. Let the smear air-dry. (The smear looks even across the entire surface)

NOTES: Does not stain the bacteria, but stains the background. The bacterial will appear clear against a stain background. Why? Because of ionic repulsion- the bacteria and the acidic stain both have negative charges.

FUNCTION: Useful for identifying and classifying bacteria. This stain allows you to classify bacteria as gram positive or gram negative. (Hans Christian Gram, 1884) Those that decolorize easily are gram negative. Those who retain the primary stain are gram positive. They stain differently because of the chemical and physical differences in their cell walls. Crystal violet is picked up within the cell, Iodine reacts with the dye in the cytoplasm to form CV-1. In gram negative cell, the decolorizing agent dissolved the outer lipopolysaccharide layer, and the CV-1 washed out through the thin layer of peptiglycon.

PROCEDURE:
1. Cover the smear with Crystal Violet for 30 seconds.
2. Gently wash off the CV by squirting water until it runs through the entire smear. 3. Cover the smear with Grams Iodine for 10 seconds.
4. Gently wash the smear with water.
5. Decolorize with alcohol.
6. Wash the smear.
7. Cover the smear with safranin for 30 seconds.
8. Wash the smear.
9. Blot dry.

NOTES: The Gram Stain should be done on young cultures of bacteria (less than 24 hours). Older cultures will not retain the primary stain and give inaccurate results.

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